The goal of this application is to understand how epithelial cells of the intestinal mucosa sort membrane glycolipids into the retrograde trafficking pathway from the plasma membrane (PM) to the endoplasmic reticulum (ER), a route exploited by various bacterial toxins and viruses. Our hypothesis is that such sorting depends on the structure/function of membrane microdomains termed lipid rafts. Cholera toxin (CT), the causative agent of Asiatic cholera, typifies the structure and function of the AB5-subunit toxins that enter host cells by traveling retrograde in this pathway on raft-associated glycolipid receptors. The molecular mechanisms of these processes are not fully understood or even completely identified. Two complementary approaches are proposed to delineate the role and physiology of lipid rafts in the retrograde trafficking pathway with respect to toxin invasion: The first approach will utilize T84 and A431 epithelial cell culture systems that reproduce the differential trafficking/sorting of CT and the related E. coli toxin LTIIb in the human intestine. Unlike CT, LTIIb is unable to partition into lipid rafts, travel retrograde to the ER, and does not induce disease in humans. We will investigate raft structure/function as it pertains to the first steps of endocytosis of CT and LTIIb as well as develop in vitro trafficking assays. These studies will test endogenous factors required for the transport of CT from early endosomes to the Golgi/ER. We aim to discover host cell molecules involved in the partitioning of CT and LTIIb into divergent pathways, thus explaining how eukaryotic cells utilize lipid rafts for subcellular organization. The second approach will use the zebrafish as a genetic model to elucidate the molecular basis for cell invasion by CT. We have recently found that zebrafish embryos demonstrate a visual and biochemical phenotype in response to CT intoxication that is both specific and complete within the population. These factors will allow for the employment of an unbiased forward genetic screen aimed at identifying mutants that demonstrate an attenuated response to CT due to a lesion in an involved gene. The relevant gene(s) from any such mutants will be identified and studied in our intestinal ceHmodelsystems. We also propose reverse genetic experiments in the zebrafish that will test specific gene products for involvement in retrograde trafficking. This,award will provide the applicant with an essential period of career development^ epithelial cell biology and genetics as well as the resources to pursue a project that has high potential for transitioning into an independent research program for the candidate. Cholera toxin is the causative agent of Asiatic cholera and results in massive seretory diarrhea. Mucosal infections, typified by cholera, are the leading cause of death in children less than 5 years old around the world. This proposed research will discover novel host cell factors involved in the pathogenesis of cholera not yet ascribed to such a role, and may lead to new approaches for treatment and prevention.